Formulation Development of Ketoconazole Ophthalmic Formulation

Varsha R. Sandhan*, S.B. Gondkar, R. B. Saudagar

Department of Quality Assurance Techniques, R.G. Sapkal College of Pharmacy, Anjaneri, Nashik-422213, Maharashtra, India

ABSTRACT:

The poor bioavailability and therapeutic response exhibited by conventional ophthalmic solutions due to rapid precorneal elimination of drug may be overcome by the use of ophthalmic gel systems. The purpose of the present study was to develop ophthalmic gel formulations of ketoconazole. Intraocular delivery of topically applied drugs such as ketoconazole is hampered by elimination of the solution due to tear turnover, so an mucoadhesive gel was formulated. Ketoconazole gels were prepared using sodium carboxymethylcellulose (NaCMC) as a mucoadhesive polymer and xanthan gum as a viscosity increasing agent. Gels were evaluated for various parameters like appearance, pH, drug content, gel strength, bioadhesion, viscosity, In-vitro drug release, isotonisity, sterility, antifungal activity, ocular irritancy and stability studies. The gel strength, bioadhesion and isotonisity shown quality parameter for ophthalmic formulation. The optimized formulation containing 1% w/v Na CMC and 0.2% w/v xanthan gum have shown 97.66% drug release up to 8 hrs. This is sufficient for antifungal activity. Diffusion studies have shown that a Korsmeyers-peppas is the best-fit model. This study found that an optimized formulation having improved viscosity and better mucoadhesive property may improve the bioavaibility of ocular administration of ketoconazole in gel form and can be alternative to the conventionally administered oral formulation and effectively used to prolong residence time.

KEYWORDS: Ophthalmic drug delivery, Bioavaibility, mucoadhesive polymer, fungal keratitis.

INTRODUCTION:

Ophthalmic drug delivery is one of the most interesting and challenging endeavors facing the pharmaceutical scientist. Eye drops are conventional ophthalmic delivery systems often result in poor bioavailability and therapeutic response, because high tear fluid turnover and dynamics cause rapid precorneal elimination of the drug. A high frequency of eye drop instillation is associated with patient non-compliance. Inclusion of excess drug in the formulation is an attempt to overcome bioavailability problem is potentially dangerous if the drug solution drained from the eye is systemically absorbed from the Nasolachrymal duct. The specific aim of designing a therapeutic system is to achieve an optimal concentration of a drug at the active site for the appropriate duration.^[1] Ophthalmic mycosis is emerging as a major cause of vision loss and morbidity, and can be life-threatening. Fungal keratitis is one of the major causes of ophthalmic mycosis. Fungal keratitis is usually characterized by a corneal epithelial defect and inflammation of the corneal stroma.

Table 1. Composition of formulation batches as per 3² factorial design

Ingredient (%)	Formulation code
	F1	F2	F3	F4	F5	F6	F7	F8	F9
Ketoconazole(w/v)	1	1	1	1	1	1	1	1	1
Sodium Carboxymethylcellulose(w/v)	0.2	0.4	1	0.2	0.4	1	0.2	0.4	1
Xanthan Gum(w/v)	0.2	0.2	0.2	0.4	0.4	0.4	1	1	1
Sodium metabisulphite(w/v)	0.02	0.02	0.02	0.02	0.02	0.02	0.02	0.02	0.02
Bezalkonium chloride(v/v)	0.01	0.01	0.01	0.01	0.01	0.01	0.01	0.01	0.01
PEG 400(v/v)	60	60	60	60	60	60	60	60	60
Purified water(v/v)	40	40	40	40	40	40	40	40	40

If untreated, fungal keratitis can lead to corneal scarring and vision loss. It is usually caused by Aspergillus, Candida, and Fusarium species.^[2] Ketoconazole is antifungal drug and has a broad spectrum of activity, including against Aspergillus, Candida, and Fusarium species. When it given orally for long term treatment of fungal keratitis it has some disadvantages. Hence topical 1% formulation is suitable for treatment of fungal keratitis. These have been reported to inhibit the progression of corneal fungal infections and were not associated with significant corneal toxicity.^[2] To increase the patient compliance and to have convenience of administration, ophthalmic gel of Ketoconazole was prepared by using mucoadhesive polymers which can be increases its residence time and subsequent bioavailability.

MATERIALS AND METHOD:

Materials

Ketoconazole was gifted by Glenmark Pharmaceuticals Ltd. (Nashik, India), Na CMC was obtained from Reliance Cellulose and Xanthan gum obtained from Signet Chemicals. All other chemicals used were of analytical grade.

Method

3² factorial design was used for composition of different formulations (Table 1.) all different formulations were prepared as per Table 2.

Table 2. Experimental Design as per 3² Factorial Design

Formulation code	Coded values
	X1	% (W/V)	X2	% (W/V)
F1	-1	0.2	-1	0.2
F2	0	0.4	-1	0.2
F3	+1	1	-1	0.2
F4	-1	0.2	0	0.4
F5	0	0.4	0	0.4
F6	^+1	1	0	0.4
F7	-1	0.2	+1	1
F8	0	0.4	+1	1
F9	+1	1	+1	1

Accurately weighed quantity of the Ketoconazole was dissolved in PEG 400. The Sodium metabisulphite was added to above mixture with continuous stirring. The Na CMC and Xanthan gum was sprinkled over of deionised water and was allowed to hydrate for 12 hours to produce a clear solution. The Bezalkonium chloride was added to the above polymer dispersion. Both drug solution and polymer dispersion were autoclaved at 121°C for 20 min. and polymer dispersion was slowly added to the drug solution under aseptic condition.

Evaluation of ophthalmic gel

Determination of clarity, p^H and drug content

The clarity was determined visually. The pH of each formulation was determined by using Digital pH meter (Digital pH meter 335). ^[3]

The drug content determined by 0.5gm of gel was taken in 100 ml beaker; in that beaker 50 ml methanol was added. Aliquot 1 ml from this solution was diluted up to 10ml with methanol to get the final concentration of 10 μg/ml. The absorbance of prepared solution was measured at 242.8 nm by using UV visible spectrophotometer. ^[4]

Compatibility Study ^{[5, 6]}

Compatibility study was carried out by using Fourier transform infrared spectrophotometer (8400 s Shimadzu). FTIR study was carried on pure drug and physical mixture of drug and polymers. Physical mixtures samples kept for 1 month at 40⁰C. The infrared absorption spectrum of Ketoconazole and physical mixture of drug and polymers was recorded with a KBr disc over the wave number 4000 to 400 cm^-1.

Rheological study ^[7]

The rheological properties of gels were determined by the Brookfield viscometer; type DV-II + PRO using spindle no.62 and 63.Viscosity of the formulations were taken at two different temperatures that is at room temperature and the 37⁰C with varying shear rate.

Measurement of the gel strength ^{[8, 9]}

A sample of 50 g of the gel was put in a 50 ml graduated cylinder. A weight of 14.33 g was placed on the gel surface. The gel strength, which is an indication for the ophthalmic gel at physiological temperature, was determined by the time in seconds required by the weight to penetrate 5 cm into the gel. All measurements were performed in triplicate (n=3). The apparatus used for measuring gel strength is shown in Fig.1

Fig.1: Gel strength measuring device (A) weights (B) device

(C) Graduated cylinder (D) gel

Bioadhesive Strength ^{[8, 9]}

“Detachment Stress is the force required to detach the two surfaces of mucosa when a formulation/gel is placed in between them”. The detachment stress was measured by using a modified analytical balance. A fresh goat corneal membrane was obtained from local slaughter house. A section of fresh cornea was cut from the goat eye and washed with Saline solution. A corneal membrane was fixed on a flat surface of object (such as bottom of vial) which was moistened with saline Solution. Another object which is having flat surface at bottom (such as vial) was used to which other corneal membrane was attached. This object was attached to the one side of pan (with cornea attached to flat surface in downward position). Formulations were placed onto a corneal membrane surface of object which was fixed in position. Then height of second object was adjusted so that corneal surface of both objects came in intimate contact. Two minute contact time was given to ensure intimate contact between tissues and formulation. Then weight was kept rising in the pan until the adjustable object get detached. The weight required to detach the ocular mucosa surfaces gave the mucoadhesive strength assessed in terms of weight (gm). The mucoadhesive strength was measured in forms of force of adhesion in Newton’s by using equation 1. All measurements were performed in triplicate (n=3).

Detachment Stress (dyne /Cm²) = m×g/A…………. (1)

Where, m= weight required for detachment of two vials in gms,

g= Acceleration due to gravity (980 cm/s²)

A= Surface area exposed.

The Apparatus for Bioadhesive study shown in Fig.2

Fig. 2: Modified Balance for Bioadhesive Study

A: Modified balance, B: Weighing pan, C: Glass vials, D: Gel, E: Corneal mucosa W: Weight

Isotonisity Evaluation ^[10]

The formulations were mixed with few drops of diluted blood on a slide. The diluted blood was prepared by using Grower’s solution and Slide was observed under microscope at 45x magnification. The shape of blood cells were compared with standard marketed ophthalmic formulation.

In-vitro Drug Release Study ^{[11, 12]}

In-vitro release study of the formulated ophthalmic gel was carried out by using diffusion cell through egg membrane as a biological membrane. Diffusion cell with inner diameter 24mm was used for the study. the formulation 0.5 gm were placed in donor compartment and Freshly prepared 100 ml artificial tear fluid (sodium chloride 0.670g, sodium bicarbonate 0.200g, calcium chloride dehydrated 0.008g,purified water q.s 100ml.) in receptor compartment. Egg membranes were mounted in between donor and receptor compartment. The position of the donor compartment was adjusted so that egg membrane just touches the diffusion medium. The whole assembly was placed on the thermostatically controlled magnetic stirrer. The temperature of the medium was maintained at 37°C ± 0.5°C. 2ml of sample is withdrawn from receiver compartment after 30 min, 1, 2, 3, 4, 5, 6, 7and 8 hrs and same volume of fresh medium is replaced. The withdrawn samples was diluted to 10ml in a volumetric flask with methanol and analyzed by UV spectrophotometer at 242.8nm.

Antifungal Activity ^{[13, 14]}

An agar diffusion method was used for the determination of antifungal activity of formulations. Inocula were prepared by suspending 1-2 colonies of Candida albicans (NCIM no. 3102) from 24 hr cultures in Sabouraud's medium into tubes containing 10 ml of sterile saline. The inoculum (0.5 ml) was spread over the surface of agar and the plates were dried at 35°C for 15 min prior to placing the formulation. The bores of 0.5 cm diameter were prepared and 20 μl samples of formulation (1% w/v) were added in the bores. After incubation at 35°C for 24 h, the zone of inhibition around the bores was measured.

Test for Sterility ^{[15, 16]}

The sterility test was carried out as per IP (1996) method. The optimized formulation was incubated for not less than 14 days at 30 -35⁰c in the fluid thioglycolate medium and at 20-25⁰c in soyabean casein digest medium to find out growth of fungi in formulation.

Ocular Irritancy Test ^{[17, 18]}

The optimized formulation was used for eye irritancy study. The protocol was approved by Institutional Animal Ethics Committee with approval no-IAEC/01.

The Modified Draize Eye Irritation: The 03 Albino rabbits weighing 1.5 to 2 kg. According to the draize test, the amount of formulation was applied to the eye is 100μl was placed into the lower cul-de-sac. Test solution was instilled in left eye and saline solution was instilled in right eye. The evaluation of ocular lesions was made at 1, 4, 24, 48,72hrs, and 1 week after administration. 3 day washing period with saline was carried out. The rabbit was observed periodically for redness, swelling, watering of the eye.

Accelerated stability studies ^[19]

The formulations were stored at room temperature and 40± 2°C with RH 60± 5% and 75± 5% respectively. The formulations were evaluated mainly for their physical characteristics at the predetermined intervals of 3 months and after 6 months like appearance/clarity, pH, viscosity and drug content.

RESULTS AND DISCUSSION:

Clarity, p^H and Drug content

On careful visual inspection against dark and white background, all the prepared ophthalmic gel formulations were found to be free from any suspended particulate matter. All the formulations were found to be clear. This also indicates that the PEG 400 can be a good solubilizing agent at 60% v/v concentration for solubilizing 1% ketoconazole in prepared formulation.

The pH of all the formulations from F1 to F9 was found to be in the range of 6.5 to 6.8 pH values of formulations shown in Table 3. Ideally, the ophthalmic solutions should possess pH in the range of 6.5-8.5, so as to minimize discomfort or excessive tear flux causing faster drainage of the instilled dose due to corneal irritation.

The percentage drug content of all prepared ophthalmic formulations was found to be in the range of 98-102%. Table 3.Therefore uniformity of content was maintained in all formulation.

Fig.3: Fourier Transform Infra-red overlay of drug with individual polymer

Table 3. Evaluation Parameters

Sr. No	Formulation code	Observed pH (±S.D.)	Gel strength (sec) (±S.D.)	Detachment stress (dyne/cm^2) (±S.D.)	Drug content (%) (±S.D.)	Cumulative Drug Release (%) (±S.D.) after 8hrs
1	F1	6.75±0.01	0.916±0.1357	1105.15±100	98.15±0.85	32.53±0.030
2	F2	6.52±0.01	1.176±0.0493	1333.29±66.71	100.04±1.000	59.31±0.69
3	F3	6.74±0.02	5.09±0.1682	1842.96±142	101.25±0.6726	97.66±0.03
4	F4	6.5±0.208	2.92±2.0344	1611.38±111.38	98.976±0.9900	30.78±0.04
5	F5	6.54±0.02	8.913±1.8170	2205.93±105	98.97±1	53.60±0.024
6	F6	6.66±0.02	4.57±0.4453	1694.08±194.08	98.17±1.0424	83.79±0.01
7	F7	6.51±0.015	30.299±0.9209	3280.81±20	100.42±1	28.57±0.025
8	F8	6.52±0.02	12.493±3.4525	2615.41±84.59	98.11±0.89	51.35±0.025
9	F9	6.83±0.04	2.66±0.3523	1335.48±104.52	98.91±2	62.97±0.015

Table 4. Viscosity of Formulations at 37⁰C

rpm	Viscosity (cps) at 37^0C
	Formulation code
	F1	F2	F3	F4	F5	F6	F7	F8	F9
0.5	550.8	3496	39832	33353	43431	39592	74864	55428	27354
1.0	529.9	3209	27234	23635	25195	25555	49070	37672	21595
1.5	516.8	2959	20956	18876	18396	18716	36072	28234	18516
2.0	505.6	2696	17936	16137	14397	15297	29934	22735	15357

Fig. 4: Viscosity profile of formulations at 37°C

Compatibility Study

Infra-red spectra of drug and polymers showed matching peaks with the drug spectra. Fig.3 The characteristic peaks of drug were also present in the spectra of all drug- polymer combinations. Which indicate that there is compatibility between drug and polymers.

Rheological study

Viscosity v/s rpm plots for all formulations shows decrease in viscosity as shear rate (rpm) was increased. As temperature was increased the decrease in viscosity was observed. Which indicate that gel has the pseudoplastic flow. Concentration of xanthan gum was a major factor affecting viscosity of formulations. In combination with Na CMC xanthan gum has shown considerable increases in viscosity when concentration of Na CMC is 0.2% w/v to 0.4% w/v. But as the concentration of Na CMC is increased from 0.2% w/v to 4% w/v and subsequently to 1% w/v the viscosity has decreased (Table 4.) which may be because of greater swelling ability of xanthan gum than Na CMC.

Gel Strength

The gel strength was found to be affected by concentrations of gelling and mucoadhesive polymers. Optimal mucoadhesive gel must have suitable gel strength so as to be administered easily and can be retained at ocular region without leakage after administration. Gel strength of all formulations showed comparable results as that of viscosity results. (Table 3.)

Bioadhesive Strength

Bioadhesive force means the force with which gels bind to ocular mucosa. Greater bioadhesion is indicative of prolonged residence time of a gel and thus prevents its drainage from cul-de-sac.

b. Blood cells with ketoconazole Formulation (F3)

c. Blood cells with Gentamycin as marketed formulation

Fig. 5: Shape of Blood cells

Fig. 6: In-vitro drug release profile of formulations

The Bioadhesion force increased significantly as the concentration of bioadhesion polymers increased. Results of this test indicate that the variable xanthan gum and Na CMC both are having effect on bioadhesive strength. (Table 3.) It shows that bioadhesive force was increased with the increasing concentration of the xanthan gum.

Isotonisity Evaluation

Isotonisity testing of formulations (F1, F5, F9 and F3) exhibited no change in the shape of blood cells (Fig.5) which reveals the isotonic nature of the formulations as compared with standard marketed ophthalmic formulation. This indicates the maintenance of tonicity in prepared formulations. Isotonisity was maintained to prevent tissue damage of eye.

In-vitro Drug Release Study

Out of nine formulations maximum release after 8 hrs was found for F3 formulation (Table 3). This indicates release of 97.66% drug available for antifungal activity of the drug.F3 formulation showed steady state release up to 8hrs which also indicates that this formulation would show better contact with biological membrane.

In-vitro drug release profile of formulations shown in Fig.6

Optimization

A 3² full factorial design was selected and the 2 factors were evaluated at 3 levels, respectively. The percentage of Na CMC (X₁) and xanthan gum (X₂) were selected as independent variables and the dependent variable was % drug release_.

Fig.7: Surface Response plot showing effect of sodium carboxymethylcellulose and xanthan gum on drug release.

The data obtained were treated using Design expert version 8.0.4.1 software and analyzed statistically using analysis of variance (ANOVA). The data were also subjected to 3-D response surface methodology to study the interaction of Na CMC (X₁) and xanthan gum(X₂) on dependent variable. The values of X₁ and X₂ were found to be significant at p <0.05, hence confirmed the significant effect of both the variables on the selected responses. From this data optimum concentration of Na CMC 1% w/v and xanthan gum 0.2%w/v was found (Fig.7).

Y1 (% CDR) = Y1 (% CDR) = 38.7576+61.1679*(A)-18.18013*(B)

From design expert version 8.0.4.1 five solutions were found in which optimum batch Na CMC 1%w/v and xanthan gum 0.2% w/v with desirability 1 was found to be optimum. From this data F3 batch was selected as optimum formulation.

Release Kinetics ^{[20, 21]}

In the present study, the drug release was analyzed by PCP Disso version v3 software to study the kinetics of drug release mechanism. The factorial design batches followed korsmeyer peppas model kinetics. The R² value of korsmeyer peppas model was found close to one. The drug release was occurred by fickian diffusion mechanism as reflected by its n value 0.1758 (n<0.5).

Antifungal Activity

The study of indicate that ketoconazole retained its antifungal efficacy when formulated as an ophthalmic gel and drug was active against selected strains of micro-organism.

Table 5. Zone of inhibition and % efficacy of formulations

Sr.no	Formulation Code	Candida albicans
		Zone of Inhibition (mm) ±SD	% Efficacy
1	Standard value	18	100
2	F1	09±1	50.00
3	F2	12.5±2.5	69.44
4	F3	18±5.65	100
5	F4	8.5±3.53	47.22
6	F5	12.5±7.77	69.44
7	F6	16.5±3.53	91.66
8	F7	04±1	22.22
9	F8	10±7.07	55.55
10	F9	14.5±7.77	80.55

F3 formulation showed 18mm zone of inhibition and 100% efficacy. (Table 5) Results obtained from antifungal activity of F3 formulation resembles to release profile of drug which indicate the dependency of antifungal activity with drug release from formulation.

Test for Sterility

There was no appearance of turbidity and hence no evidence of fungal growth when optimized formulation was incubated for not less than 14 days at 30⁰C to 35⁰C in case of fluid thioglycolate medium and at 20⁰C to 25⁰C in case of soyabean-casein digest medium. The preparations examined, therefore, passed the sterility test.

Ocular Irritancy Test

The results of the ocular studies indicate that the formulation F3 was non-irritant and no ocular damage or abnormal clinical signs were visible.

Table 6. Observations of Ocular Irritancy Study

Time	Redness		Swelling		Watering
	Rabbits
	1,2,3	1,2,3	1,2,3	1,2,3	1.2,3	1,2,3
	Right Eye	Left Eye	Right Eye	Left Eye	Right Eye	Left Eye
At the time of installation(0hr)	0	1	0	0	1	1
10min	0	0	0	0	0	0
1hr	0	0	0	0	0	0
4hr	0	0	0	0	0	0
24hr	0	0	0	0	0	0
48hr	0	0	0	0	0	0
72hr	0	0	0	0	0	0
1 week	0	0	0	0	0	0

CONCLUSION:

This solubility enhanced ketoconazole 1% ophthalmic gel formulation fulfills all necessary parameters required for ophthalmic use. This optimized formulation having improved viscosity and better mucoadhesive property may improve the bioavaibility of ocular administration of ketoconazole in gel form and can be alternative to the conventionally administered oral formulation.

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Received on 15.07.2013

Modified on 17.08.2013

Accepted on 23.08.2013

© A&V Publication all right reserved

Research Journal of Pharmaceutical Dosage Forms and Technology. 5(6): November-December, 2013, 303-310